Cardiovascular disease is the sum total of a series of molecular and cellular responses to stressful stimuli. An increase in expression of stress proteins such as heat shock protein 70 (Hsp70) is a common cellular response to oxidative stress, cytokine release and ischemia-reperfusion. Its expression is correlated with the protection of cardiomyocytes and the whole heart in the event of these environmental insults. The mechanisms by which Hsp70 exerts its protective effects are as yet undetermined, but may involve its role as a molecular chaperone and its association with protein degradation pathways. Hsp70's involvement in these processes is regulated both transcriptionally and by co-chaperones. This proposal involves a recently described negative regulator of Hsp70/Hsc70 activity, carboxyl terminus Hsc70-interacting protein (CHIP). The objective of this proposal is to determine the effect of CHIP on cellular pathways/processes influenced by Hsp70 expression with regards to CHIP's ability to alter Hsp70's activity. CHIP expression will be manipulated in cardiomyoblasts (H9c2) and myocytes (C2C12) using a retroviral-mediated gene transfer technique. Overexpression of CHIP will be the focus of investigations regarding the involvement of CHIP in cell pathways known to be affected by Hsp70 and of particular relevance to cardiovascular disease. Cells will be treated with known inducers of oxidative stress and apoptosis. Production of cytokines, NF-kB activity, and specific activators (SAPK/JNK) and effectors (caspase-activated enzymes) in the apoptotic cascade will be analyzed. This work will determine if CHIP is involved in mediating Hsp70's protection in response to stressful stimuli which are known inducers of atherogenesis and cardiovascular disease.